The present invention relates to a microbial process for the preparation of compositions containing acetophenone. In a further aspect, the present invention relates to products produced by the microbial process.
In a still further aspect, the present invention relates to organoleptic uses of said products.
In today""s market, it is frequently desirable to identify flavor components of food items and other consumer products as containing xe2x80x9cnatural flavorsxe2x80x9d or xe2x80x9cnatural ingredients.xe2x80x9d It is generally recognized in the industry that a flavor compound having been prepared by microbial processes can be designated as a natural product and therefore have an important place in the commercialization of products containing them. As a result, the industry has devoted considerable time and effort to develop methods for the production of ingredients for food and other consumer items and in particular for the production of acetophenone which can be called xe2x80x9cnatural.xe2x80x9d
Thus, as an example of such prior developments a method for preparing acetophenone utilizing a mutant microorganism of the microbial genus Pseudomonas is disclosed in U.S. Pat. No. 4,871,688. The nutrient medium contains a cinnamate compound as a carbon source. However, the procedure shown therein utilizes an unknown microorganism with the deposit number ATCC 53716 which process is not shown to be broadly applicable to other types of microbes. In a study by the same inventors in APPLIED AND ENVIRONMENTAL MICROBIOLOGY of March 1990, pages 623-627, there is disclosed that an unclassified Pseudomonas species can be used to form acetophenone from cinnamic acid.
A number of studies have shown that acetophenone can be metabolized by certain species of Arthrobacter and Nocardia in Eur. J. Biochem., 86, 175-186 (1978). There are also some studies showing liquidation of acetophenone by certain bacterial species as shown in Biosci. Biotech. Biochem., Vol. 59, (12), pages 2324-2325, 1955. Other studies involving bacterial degradation and metabolism of the acetophenone are found in APPLIED AND ENVIRONMENTAL MICROBIOLOGY, September 1979, pages 514-520, May 1987, pages 1103-1112 and December 1990, pages 3678-3685. Oxidative production of acetophenone is reported by Ohta, et al. in Agric. Bio. Chem., Vol. 48 (6), 1509-1516, 1984.
Conversion of 1-phenethyl alcohol to acetophenone utilizing a microbe is shown by Lee, et al. in APPLIED AND ENVIRONMENTAL MICROBIOLOGY, September 1996, pages 3101-3106. Ethylbenzene converted to 1-phenethyl alcohol and acetophenone utilizing fungi is shown by Holland, et al. in CAN. J. CHEM., Vol. 65, 1987, page 502 et seq.
Use of Arthrobacter for transforming n-hexadecane to ketones is shown by Klein, et al., APPLIED MICROBIOLOGY, May 1969, pages 676-681.
Production of ketones and alcohol using Arthobacter with metabolism of cyclohexaneacetic acid is described by Ougham, et al. in the JOURNAL OF BACTERIOLOGY, June 1982, pages 1172-1182.
Metabolism of acetophenone is shown by Cripps in the Biochem J. (Great Britain), 1975, Vol. 152, pages 233-241.
The Arthrobacter genus of bacteria is well known and described in the literature.
Such prior art methods are said to be economically attractive but there is a constant need for improvement of yields and conversion which is addressed in this invention.
In the flavor and fragrance art, the need has arisen for the development of more efficient production of acetophenone which has heretofore been found to be useful and necessary in the creation of flavor formulations used in augmenting or enhancing the aroma or taste of such items as foodstuffs, chewing gums and toothpastes, and which is also useful in augmenting or enhancing the aroma of perfume compositions such as colognes, perfumed articles in either solid or liquid state as, for example, ionic, cationic, nonionic or zwitterionic detergents, perfumed polymers, fabric softener compositions, fabric softener articles, hair preparations, cosmetic powders and the like.
It is therefore an object of the present invention to provide a new and improved method for preparing acetophenone by a fermentative process to result in a natural ingredient suitable for a wide variety of purposes.
It is a further object of the present invention to produce acetophenone in a more efficient manner to obtain a higher yield and greater conversion than prior known biological methods.
The above and other objects and features of the invention are obtained in accordance in the present invention by carrying out a fermentation process using selective reaction techniques to produce and recover acetophenone which is useful for its organoleptic properties in augmenting or enhancing the aroma or taste of consumable materials such as foodstuffs, chewing gums, toothpastes, perfume compositions, colognes and perfumed articles such as solid or liquid detergents, perfumed polymers, fabric softener compositions, fabric softener articles, cosmetic powders, hair preparations and the like.
Acetophenone is represented by structure: 
The fermentation reaction to produce acetophenone according to the invention is carried out under oxidative conditions by preparing an aqueous inoculum nutrient medium including a selected bacterium species in a first liquid phase, and a second liquid phase containing a source of cinnamic acid, either as the free acid or a salt thereof in the form of a cinnamate. Cinnamic acid is represented by the structural formula: 
Alkali metal salts such as sodium cinnamate have also been found to yield good results for this fermentation reaction.
The inoculum medium containing the usual buffers, mineral salts, yeast extract and the like is an aqueous, generally neutral pH system and is sterilized. Then the aqueous material medium is inoculated with the selected bacterium species and incubated for a sufficient period of time with sufficient vigorous agitation to form a viable culture.
The second phase is the acetophenone production system containing essentially the same components as the inoculum medium. This is also sterilized.
The first liquid phase and the second liquid phase are then mixed together with agitation to form a single-phase system and permitted to undergo further incubation. Then the source of cinnamic acid is added in increments at a rate sufficient to maintain a concentration of cinnamic acid equivalent to at least 5 g/l[e.g. 5-10 g/l]. The broth is initially neutral at approximately pH=7. At the conclusion of the fermentation, the broth is acidified to pH 4. Following recovery, the product is obtained in a yield of at least 90% to 99+%.
A further feature of the present invention resides in the products produced by the present invention characterized by the GLC profiles which accompany this application.
Still further, another feature of the invention resides in the flavor and fragrance compositions containing the acetophenone produced by the present invention.